Morphologically accurate biomechanical models of the intervertebral disc anulus fibrosus (AF) require precise knowledge of its lamellar architecture; however, available methods of assessment are limited by poor spatial resolution or the destructive nature of the technique. In a novel approach, diffusion tensor microscopy was used in this study to characterize the microstructure of excised porcine AF samples. Results show diffusion in the AF to be anisotropic. The orientations of anisotropy exhibit a layered morphology that agrees with light micrographs of the corresponding samples, and the behavior of the orientation angles is consistent with the known AF collagen fiber architecture. A static magnetic field-dependent relaxation anisotropy was observed in the AF, which has methodological implications for magnetic resonance (MR) imaging of ordered collagenous tissues. These findings present MR diffusion tensor microscopy as a potentially valuable tool to assess quantitatively and nondestructively water diffusion anisotropy and lamellar structure of the intervertebral disc AF.